Colorectal cancer is the third leading cause of cancer mortality in both men and women in the United States. Resistance to fluoropyrimidine-based (e.g. 5-fluorouracil/Oxaliplatin/Leucovorin) chemotherapy is one of the major reasons for the failure of treating advanced colorectal cancer. The lack of ability for tumor cells to undergo apoptosis after genotoxic stress is one of the key contributors to such resistant mechanism. Mounting evidence has demonstrated that non- coding microRNAs (miRNAs) are crucial regulators of gene expression, in particular, under acute genotoxic stress. However, there is still limited knowledge about the role of miRNAs in apoptosis in colorectal cancer. Our recent studies discovered a novel mechanism of miR-129 mediated apoptosis by suppressing the expression of anti-apoptotic protein Bcl2. In addition to Bcl2, miR-129 also suppresses the expression of 5-fluorouracil (5-FU) target protein thymidylate synthase (TS) and cell cycle control gene E2F3. Ectopic expression of miR-129 restored apoptosis in colon cancer cell lines by suppressing Bcl2 and sensitized colon cancer cells to 5- FU treatment both in vitro and in vivo. We further demonstrated that the loss of miR-129 expression in colorectal cancer patients is a critical event during disease progression. More importantly, we were able to reverse the chemoresistance in colon cancer stem cells with miR- 129. Based on these, it is important to further investigate the regulatory mechanism of miR-129 and its clinical relevance and impact in colorectal cancer. In the proposed project, we will test the hypothesis that miR-129 is indeed a tumor suppressor of colorectal cancer and a novel modulator of chemosensitivity to 5-FU based treatment. In Specific Aim 1, we will test the hypothesis that modulating miR-129 expression can be an effective approach to restore apoptosis, cell cycle control, and reverse chemoresistance in colon cancer stem cells. Specific Aim 2, we will test the clinical relevance of miR-129 in colorectal cancer using various colon cancer mouse models and patient specimens. Specific Aim 3, we will test the hypothesis the miR-129 is a tumor suppressor by fully understanding the mechanism of its major targets and pathways. The proposed investigation will provide further understanding of miR-129 in colorectal cancer progression and response to 5-FU based chemotherapy. It has the potential to develop novel therapeutic approaches to enhance 5-FU efficacy to benefit patients.